medRxiv. 2026 May 25. pii: 2026.05.22.26353710. [Epub ahead of print]
Jorge Gomez Tejeda Zanudo,
Busem Binboga Kurt,
Allison Frangieh,
Alice M Barkell,
John Navarro,
Lan Ngo,
Ayesha Mohammed-Abreu,
Verena M Prade,
Ina Bisha,
Sudhanshu Abhishek,
Beom-Jun Kim,
Melissa Hughes,
Karla E Helvie,
Joanna Baginska,
David J Clark,
Markus Schick,
Ross J Hill,
Tari A King,
Elizabeth A Mittendorf,
Marlon Rebelatto,
Eric P Winer,
Sara M Tolaney,
Bruce E Johnson,
Danielle Carroll,
Maurizio Scaltriti,
Nancy U Lin,
Elza C de Bruin,
Ana C Garrido-Castro.
Introduction: With recent approvals of multiple targeted therapies for triple-negative breast cancer (TNBC), including antibody-drug conjugates and immunotherapy in biomarker-selected populations, it is critical to define the temporal evolution of cell-surface target expression from early-stage to metastatic disease, the co-expression patterns across these markers, and optimal quantification methodologies. Here we report biomarker expression profiles measured by multi-omics and pathology-based platforms in patients with TNBC using a large cohort of matched longitudinal tumor samples.
Methods: Patients who underwent neoadjuvant chemotherapy (NAC) for stage I-III TNBC or were diagnosed with any stage TNBC and developed metastatic recurrence were retrospectively identified from an institutional database and prospective research metastatic biopsy protocol. Tumor samples from diagnosis (DX), residual disease (RD) post-NAC (if applicable), and metastasis/recurrence (MR) were collected. Quantification of HER2, TROP2, and PD-L1 expression was performed by immunohistochemistry (IHC), whole-exome sequencing, transcriptome sequencing, and targeted mass spectrometry (MS). For HER2, TROP2, and stromal tumor-infiltrating lymphocytes (sTILs), both manual pathologist assessment and computational pathology quantification were obtained. HER2 status was categorized as HER2-0 or HER2-low by local (L-IHC) and central (C-IHC) review, TROP2 status was defined as low (H-score <100), medium (H-score 100-200) or high (H-score >200), and PD-L1 as low (tumor area positivity, TAP <5%) or high (TAP ≥5%). Pathologist-assessed sTILs were classified as low (<10%), medium (≥10% and <40%) or high (≥40%). Biomarkers were compared between primary (DX/RD) and MR, and between pre- vs post-NAC (DX-RD) samples. Correlations between markers, quantification methods, inferred PAM50 subtype, and clinical variables of interest were evaluated.
Results: A total of 359 samples from 110 patients with TNBC with data available from at least one platform were included in the analysis. HER2-low prevalence at DX, RD, and MR was: 51% (50/98), 40% (21/53), and 27% (16/60); TROP2 high/medium was 90% (47/52), 91% (42/46), and 88% (28/32); PD-L1-high was 51%, 50%, and 38% (9/24); and sTILs-high/medium was 88% (59/67), 80% (40/50), and 49% (17/35), respectively. While TROP2-high/medium vs low remained stable over time, HER2 IHC and sTILs significantly decreased from DX/RD to MR samples, both at the cohort-level (HER2, p=0.0081; sTILs, p=4.6x10e-5) and longitudinal patient-level (HER2, p=0.030; sTILs, p=0.0077), with a similar decreasing trend for PD-L1 that did not reach statistical significance. HER2 concordance (0 vs low) between L-IHC and C-IHC was 78% (91/116). ERBB2 , TACSTD2 and CD274 mRNA expression were significantly correlated with IHC protein levels, though only TACSTD2 had limited overlap in distribution of gene expression between high/medium vs low groups. Strong correlation between protein membrane staining intensity from computational pathology, protein expression measured by MS, and pathologist-assed IHC was observed across all biomarkers tested by each method. In comparisons between biomarkers, pathologist-assessed PD-L1 IHC and sTILs were significantly correlated (p=0.0001); 94% (51/54) of PD-L1-high tumors were classified as sTILs high/medium. PAM50 subtype was not significantly correlated with time point or biomarker status, although there was a trend toward more HER2-enriched tumors in HER2-low (20%, 5/25) vs HER2-0 (6%, 3/52) (p=0.086). Across biomarkers and clinical variables, an association between age and sTILs was observed (p=0.038, FDR=0.42) due to a decrease in sTILs high/medium tumors with age, primarily driven by post-treatment (RD/MR) but not DX samples.
Conclusions: Multi-platform and multi-omics profiling in this large unique cohort of longitudinal TNBC samples revealed distinct patterns of expression and dynamic changes of key biomarkers of interest for targeted therapies. Given variability with manual IHC scoring, improved methods for quantification of expression may help optimize treatment selection in an individualized manner.